Several emerging areas of biotechnology, such as molecular transduction in biosensing and surface-mediated bioseparations, rely on the function of proteins immobilized in a thin film at a solid-liquid interface. It is well recognized that the structure and function of an interfacial protein film are dependent on the physical and chemical properties of the interface, but these relationships are not well understood, in part because studying protein films in situ is technically difficult. Developing a fundamental understanding of these relationships is a prerequisite to developing methodologies designed to organize proteins into macroscopically oriented, stable, and bioactive planar arrays for use in molecular device technologies. The proposed research addresses two hypotheses in this area: 1) Ordered, asymmetric, bilayer protein films can be assembled at a solid- liquid interface using a step-wise combination of site-directed, covalent bonding and biospecific, noncovalent binding to appropriately functionalized substrate surfaces. The distribution of molecular orientations in the outermost protein layer is dependent on the physical and chemical properties of the preceding layer(s) and the substrate surface. 2) Native protein conformation and biofunction in the outermost protein layer may be perturbed by the immobilization process; the degree of perturbation is also dependent on the physical and chemical properties of the preceding layer(s) and the substrate surface. Mono and bilayer protein films will be assembled on functionalized substrates using a step-wise combination of site-directed, covalent bonding and biospecific binding strategies (Fig. 1). Yeast cytochrome c (cyt c) will form the outermost layer; the intrinsic heme group will be used as spectral probe of molecular orientation, function, and conformation. Streptavidin will form the inner layer of bilayer films; monolayer films will serve as controls for bilayer films. The molecular orientation distribution in the cyt c layer will be measured using a unique combination of in situ polarized reflection techniques, planar integrated optical waveguide-attenuated total reflection (IOW-ATR) spectrometry and total internal reflection fluorescence (TIRF) spectroscopy. Visible absorbance and resonance Raman spectroscopies will be used to study conformation and function (i.e., redox activity) in the cyt c layer, and to make comparisons to dissolved cyt c. Planar waveguide-based version of these techniques will provide the required monolayer sensitivity. The effect of substrate properties on macroscopic order, conformation, and function will be assessed by comparing films assembled on functionalized LB films and covalently modified glasses. This research addresses both the preparation of protein film assemblies and the development of analytical techniques appropriate to characterize them, because these endeavors are necessarily coupled. The results should produce a firmer understanding of relationships between the structure of hydrated protein films and their macroscopic properties, which should aid in protein film design and applications.